Our overall goal is to define the molecular/physiologic environment in the breast epithelium which permits the development of sporadic breast cancer, particularly as it relates to estrogen exposure, and to translate this knowledge into preventive strategies. Our work so far has demonstrated that the breasts of women with breast cancer (cases) over- express estrogen receptor alpha, relative to the breasts of women without breast cancer (controls). Further, we have evidence suggestive of a failure of autologous down-regulation of ER alpha by estradiol in high risk breasts. These data were developed using fresh-frozen normal breast tissue from women undergoing breast surgery,a nd are not easily reproducible for this reason. We now propose a confirmatory study using a fresh case-control data set of 400 women with paraffin embedded breast tissue retrieved from Department of Pathology archives. This data set has already been assembled, and includes detailed breast cancer risk data and information on menstrual cycle dates and exogenous hormone use. We will use this group of women to test the hypotheses that 1) ER alpha is expressed more frequently and at higher levels in normal breast epithelium of cases: 2) proliferative rates (i.e. Ki-67 labelling) are higher and 3) apoptotic rates (i.e. TUNEL positive cells) are lower in the breast epithelium of breast cancer cases, when compared to benign disease controls. These analyses will test the widely held concept that estrogen contributes to breast cancer etiology via increased proliferation and will provide crucial new information regarding the importance of disordered cell death. Finally, epidemiological studies examining breast epithelial characteristics of premenopausal women are badly hampered by the lack of menstrual cycle data, since cellular events and protein expression fluctuate with menstrual cycle phase. We will test a method of categorizing menstrual cycle phase using histologic assessment of breast epithelial samples which, if together, these studies will provide a framework for future examinations of breast epithelial biology in the cancer-prone breast, and point to pathways that can be disrupted or promoted for breast cancer prevention. The ability to retrospectively assess menstrual cycle phase will lead to the ability to utilize large banks of paraffin embedded tissue for similar studies in pre-menopausal women.